Self-renewal is essential for the homeostasis and lifetime maintenance of many organ systems. The process of self-renewal is carried out by rare populations of adult stem cells whose key features are multilineage potential and repopulating capacity. Blood cell development is driven by the successive restriction of cell fate as multipotent hematopoietic stem cells (HSCs) give rise to all mature red and white blood cell types. Current models suggest that the accumulation of cell-type specific patterns of DNA methylation correlates with cell fate specification, with the pattern of marks both creating a record of a cell's lineage and locking that cell into a particular fate due to the innate heritability of such marks. We propose to produce genome-wide reference maps of DNA methylation throughout the human and mouse genomes during HSC differentiation. Our studies will compare the state of the murine and human methylomes in purified populations of long-term repopulating HSCs. We will assess lineage-specific patterns of marks in the earliest progenitor cells for which fate is phenotypically specified, and follow the subsequent remodeling of the epigenome through blood cell maturation. This study will provide the first comprehensive examination of the state of the epigenome through a well characterized developmental cascade. Though comparative studies in two distinct models, we hope to separate the key events that drive fate specification and restriction decisions from those that might simply correlate with cell differentiation. PUBLIC HEALTH RELEVANCE: The differentiation of long-term hematopoietic stem cells into mature red and white blood cells is one of the best studied developmental processes in animals. As more primitive cells turn into more differentiated and specialized cell types, accompanying changes in the epigenetic state of the genome are thought to reinforce these decisions. We propose to examine the state of the epigenome in both stem cells and mature cells within the hematopoietic system in humans and mice. In this way, we will reveal general principles of cell fate determination and understand how cells become increasingly restricted in their potential. These studies may also reveal strategies to create specific types of hematopoietic cells for therapeutic purposes.